Journal of Steroid Biochemistry & Molecular Biology xxx (2015) xxx–xxx

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Review

Novel analogs of 1,25-dihydroxyvitamin D2 combined with a plantpolyphenol as highly efficient inducers of differentiation in humanacute myeloid leukemia cells

Matan Nachlielya, Ehud Sharonya, Andrzej Kutnerb, Michael Danilenkoa,*aDepartment of Clinical Biochemistry and Pharmacology, Ben Gurion University of the Negev, Beer Sheva 84105, IsraelbDepartment of Pharmacology, Pharmaceutical Research Institute, Warsaw 01-793, Poland

A R T I C L E I N F O

Article history:Received 15 June 2015Received in revised form 2 September 2015Accepted 8 September 2015Available online xxx

Keywords:Acute myeloid leukemiaAnalogs of 1,25-dihydroxyvitamin D2

Vitamin D receptorCell differentiationCarnosic acid

A B S T R A C T

1a,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is known to act as a powerful differentiation inducer invarious types of cancer cells, including acute myeloid leukemia (AML) cells. However, supraphysiologicalconcentrations of 1,25(OH)2D3 required to induce terminal maturation of AML cells can cause lethalhypercalcemia in vivo. Here we characterized the differentiation-inducing effects of novel double-pointmodified analogs of 1,25-dihydroxyvitamin D2 [1,25(OH)2D2], PRI-5201 and PRI-5202 [Pietraszek et al.(2013) Steroids, 78:1003–1014], on HL60, U937 and MOLM-13 human AML cells in comparison with theirdirect precursors (PRI-1906 and PRI-1907, respectively) and 1,25(OH)2D3. The results demonstrated thefollowing order of potency for the tested compounds: PRI-5202 > PRI-1907 > PRI-5201 > PRI-1906 � 1,25(OH)2D3, as determined by measuring the expression of cell surface markers of myeloid differentiation.Particularly, the sensitivity of different AML cell lines to PRI-5201 and PRI-5202 was 3-15-fold and13-50 fold higher, respectively, compared to that of 1,25(OH)2D3. Importantly, all the analogs tested at0.25–1 nM concentrations retained the ability of 1,25(OH)2D3 to cooperate with the rosemary polyphenolcarnosic acid, which strongly potentiated their prodifferentiation activity in a cell type-dependentmanner. These synergistic effects were associated with increased induction of the vitamin D receptor(VDR) protein expression. However, surprisingly, carnosic acid was able to significantly enhance only 1,25(OH)2D3-induced transactivation of the direct repeat 3 (DR3)-type vitamin D response element (VDRE),whereas no such cooperation was seen with 1,25(OH)2D2 analogs. Furthermore, dose-response analysisrevealed that 1,25(OH)2D3 was more efficacious than the analogs in inducing VDRE activation. Thissuggests that the superior prodifferentiation activity of the analogs, as compared to 1,25(OH)2D3, may bedue to their potential for enhanced activation of the differentiation-related VDRE(s) that differ from theDR3-type element tested in this study. Collectively, the results demonstrate that the new double-pointmodified 1,25(OH)2D2 analogs are much stronger inducers of myeloid differentiation than 1,25(OH)2D3

and that their efficacy can be further enhanced by combination with plant polyphenols. Thesecombinations warrant their further mechanistic and translational exploration in AML and other types ofcancer.

ã 2015 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.1. Chemicals, antibodies and plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.2. Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3. Determination of cell proliferation, viability and differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

* Corresponding author at: Department of Clinical Biochemistry and Pharmacol-

Contents lists available at ScienceDirect

Journal of Steroid Biochemistry & Molecular Biology

journal homepage: www.else vie r .com/locate / j sbmb

ogy, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O. Box 653,Beer-Sheva 84105, Israel. Fax: +972 8 640 3177.

E-mail address: misha@bgu.ac.il (M. Danilenko).

http://dx.doi.org/10.1016/j.jsbmb.2015.09.0140960-0760/ã 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: M. Nachliely, et al., Novel analogs of 1,25-dihydroxyvitamin D2 combined with a plant polyphenol as highlyefficient inducers of differentiation in human acute myeloid leukemia cells, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.jsbmb.2015.09.014

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2.4. Preparation of whole cell lysates and western blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.5. Transient transfection and reporter gene assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.6. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.1. Comparative effects of a series of 1,25(OH)2D2 analogs on cell differentiation in different AML cell lines . . . . . . . . . . . . . . . . . . . . . . . 003.2. Effects of 1,25(OH)2D3 and 1,25(OH)2D2 analogs on the growth of AML cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3. Enhancement of the antileukemic effects of 1,25(OH)2D2 analogs by carnosic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.4. Effects of VDDs and carnosic on the vitamin D receptor levels and activity in AML cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction

Acute myeloid leukemia (AML) is the most common acuteleukemia in adults and is characterized by a block of terminaldifferentiation of hematopoietic progenitors at early stages ofmyelopoiesis. This results in the accumulation of highly prolifer-ative leukemic blasts in the bone marrow which disturbs normalhematopoiesis. Among the patients with newly diagnosed AML,20–40% individuals do not fully respond to standard therapy withthe cytotoxic drugs cytarabine and daunorubicin while 50–70%patients who achieve complete remission are expected to relapsewithin 3 years, and only about 10% of these patients will survive for5 years [1]. Furthermore, elderly patients with AML are oftenineligible for this treatment due to its toxicity and comorbidities,and outcomes for these patients are particularly poor [2]. Despite anumber of experimental drugs developed for the therapy of AMLmost have failed in clinical trials. Except for gemtuzumabozogamicin that has been recently withdrawn from the market,no new agent has yet been approved for AML in the last 40 years[3]. Hence the appeal of new sources for novel antileukemic drugsthat may effectively and specifically target AML cells.

Differentiation therapy is an alternative AML treatment, basedon the induction of leukemic blasts to mature beyond thedifferentiation block. The differentiation inducer all-trans retinoicacid has proven extremely valuable in the treatment of onesubtype of AML, acute promyelocytic leukemia (APL) [4]. However,APL accounts for only �10% of AML, and no differentiation therapyis currently available for other subtypes of AML. Vitamin Dderivatives (VDDs), such as 1,25(OH)2D3, the hormonal form of

Fig. 1. Structures of the double point-modified analogs (PRI-5201 and PRI-5202) and theand D2 are shown for comparison.

Please cite this article in press as: M. Nachliely, et al., Novel analogs of 1,2efficient inducers of differentiation in human acute myeloid leukemia cejsbmb.2015.09.014

vitamin D, and its synthetic low-calcemic analogs are known toregulate multiple cell events including cell proliferation, survival,differentiation, and immune responses [5,6]. The demonstration ofmarked antiproliferative and prodifferentiation effects of VDDs onAML cell lines and patient-derived leukemic blasts has suggested apotential therapeutic significance of these agents [6,7]. However,hypercalcemia induced by supra-physiological concentrations ofVDDs still remains the major limiting factor for their clinicalapplication [8].

A growing body of research indicates that combination strategyfor VDD-based cancer therapy may prove more effective thanmonotherapy with these agents [6,9]. Preclinical studies in AMLcells have shown that VDDs can potentiate growth arrest andcytotoxicity induced by chemotherapeutic agents [10,11]. On theother hand, various compounds, such as differentiation inducers(e.g., ATRA [9,12]), epigenetically active drugs (e.g., 5-aza-20-deoxycytidine [13]), and anti-inflammatory agents [14,15] werefound to enhance VDD-induced cell differentiation. Furthermore,we and others have shown that plant polyphenols, such as carnosicacid [16,17], curcumin [18,19] and silibinin [19,20], markedlypotentiate the differentiation-inducing effects of near physiologi-cal concentrations of 1,25(OH)2D3 on AML cell lines and patient-derived leukemic blasts. We have also demonstrated that carnosicacid can synergistically enhance cell differentiation induced bylow-calcemic 1,25(OH)2D3 analogs [21,22]. The latter findings mayhave clinical implications for the low-toxic combination differen-tiation therapy of AML.

We have previously reported the synthesis and evaluation ofPRI-1906 and PRI-1907, the analogs of 1,25-dihydroxyvitamin D2

ir respective precursors (PRI-1906 and PRI-1907). Natural active forms of vitamin D3

5-dihydroxyvitamin D2 combined with a plant polyphenol as highlylls, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.

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(1,25(OH)2D2) with increased ability to induce differentiation ofAML cells compared to 1,25(OH)2D3 [23,24]. Most recently we havesynthesized the analogs, modified in the two distinct part of thevitamin D molecule (double point-modified analogs) [25].Particularly, in the structures of PRI-5201 and PRI-5202 (Fig. 1)we combined the optimized length and unsaturation level of theside-chain of PRI-1906 and PRI-1907 and a very effective 19-normodification [26]. In the present study, we characterized for thefirst time the anticancer effects of these novel compounds in vitro,employing cultured leukemia cell lines HL60, U937 and MOLM-13 that represent different subtypes of AML. The resultsdemonstrated superior differentiation-inducing effects of PRI-5201 and PRI-5202 compared to the other compounds tested. Inaddition, the effects of all the tested 1,25(OH)2D2 analogs at theirlow concentrations were potentiated by carnosic acid, which wasselected for the combination studies due to its previously reportedability to markedly enhance VDD-induced differentiation of AMLcells [16,17,21,22].

2. Materials and methods

2.1. Chemicals, antibodies and plasmids

Reference 1,25(OH)2D3 and analogs PRI-1906, PRI-1907, PRI-5201 and PRI-5202 were synthesized at the Chemistry Departmentof the Pharmaceutical Research Institute (Warsaw, Poland).Carnosic acid (>98%) was obtained from Nanjing Chemlin ChemicalIndustry Co. (Nanjing, China). Antibodies against VDR (C-20), RXRa(D-20) and calregulin (H-170) were purchased from Santa CruzBiotechnology (Dallas, TX). The VDRE � 6-Luc reporter constructcontaining a 6-fold direct repeat 3 (DR3) sequence [27] was kindlyprovided by Dr. L.P. Freedman (Memorial Sloan-Kettering CancerCenter, New York, NY, USA). Renilla luciferase expression construct(pRL-null vector) was purchased from Promega (Madison, WI, USA)and served as an internal transfection standard.

Fig. 2. Enhanced differentiation-inducing and cell type-specific antiproliferative effectagents or vehicle (�0.2% ethanol) for 96 h. (A) The expression of CD14 and CD11b was detassay. The data are the means � SD (n = 3).

Please cite this article in press as: M. Nachliely, et al., Novel analogs of 1,2efficient inducers of differentiation in human acute myeloid leukemia cejsbmb.2015.09.014

2.2. Cell culture

HL60 myeloblastic leukemia cells (ATCC-CCL-240) wereobtained from Dr. Rachel Levy (Ben-Gurion University of theNegev, Beer Sheva, Israel). U937 promonocytic leukemia cells(ATCC-CRL-1593.2) were purchased from American Type CultureCollection (Rockville, MD). MOLM-13 monocytic leukemia cells(ACC 554) were purchased from Leibniz Institute DSMZ-GermanCollection of Microorganisms and Cell Cultures (Braunschweig,Germany). Cells were grown in RPMI 1640 medium supplementedwith 10% FCS, penicillin (100 U/ml), streptomycin (0.1 mg/ml) and10 mM HEPES (pH 7.4) in a humidified atmosphere of 95% air and5% CO2 at 37 �C.

2.3. Determination of cell proliferation, viability and differentiation

Cells were seeded at 5 �104 cells/ml and treated with testagents or vehicle (�0.2% ethanol) for 96 h. Cell numbers andviability were determined on the basis of the trypan blue exclusionassay by enumerating in Vi-Cell XR cell viability analyzer (BeckmanCoulter Inc., Fullerton, CA, USA). The number of viable (trypanblue-impermeable) cells was counted directly, and cell viabilitywas calculated as the percentage of viable cells relative to the total(viable + dead) cell count. Aliquots of 5 �105 cells were harvested,washed with PBS and incubated for 45 min at room temperaturewith 0.3 mL MO1-FITC and 0.3 mL MY4-RD1 (Beckman Coulter) todetermine the expression of myeloid surface antigens CD11b andCD14, respectively, as described previously (e.g., [16]).

2.4. Preparation of whole cell lysates and western blotting

Cells were seeded at 1 �105 cells/ml and incubated with testagents or vehicle (�0.2% ethanol) for 48 h. Briefly, cells were lysedin 1% Triton X-100-containing buffer, subjected to SDS-PAGE andimmunoblotting as described previously [17]. The VDR and RXRaantibodies were used at 1:200 and 1:1000 dilutions, respectively.

s of PRI-5201 and PRI-5202 on AML cells. Cells were incubated with the indicatedermined by flow cytometry. (B) Cells were enumerated by the trypan blue exclusion

5-dihydroxyvitamin D2 combined with a plant polyphenol as highlylls, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.

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Calregulin (1:500) was used as the internal loading control. Theprotein bands were visualized using the Western LightningTM

Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Inc.,Boston, MA). The optical density of each band was determinedusing the Image Quant LAS 4000 system (GE Healthcare LifeScience).

2.5. Transient transfection and reporter gene assay

U937 cells were transiently co-transfected with the VDRE�6-luciferase reporter plasmid (0.8 mg) and Renilla luciferase vector(0.2 mg) using JetPEI Reagent (POLYplus-Transfection, IllkirchCedex, France), as described previously [17]. Transfected cellswere exposed to test agents for 24 h followed by measurement ofluciferase activity using the Dual Luciferase Reporter Assay system(Promega). The data are presented as the normalized ratios offirefly luciferase to Renilla luciferase activity (relative lumines-cence units, RLU).

2.6. Statistical analysis

Experiments were repeated at least three times. EC50 values forthe differentiation-inducing effects of VDDs were determined bynonlinear regression analysis of dose-response curves (variableslope; three or four parameters). Two compounds (A and B) wereconsidered to show synergy in the particular experiment if theeffect of their combination (AB) was larger than the sum of theirindividual effects (AB > A + B), the data being compared aftersubtraction of the respective control values from A, B, and AB [16].The values are reported as the means � SD. The significance of thedifferences between treatments was estimated by unpaired, two-tailed Student’s t-test. P < 0.05 was considered statisticallysignificant. All statistical analyses were performed with theGraphPad Prism 6.0 program (Graph-Pad Software, San Diego, CA).

3. Results and discussion

3.1. Comparative effects of a series of 1,25(OH)2D2 analogs on celldifferentiation in different AML cell lines

We first determined the differentiation-inducing effects ofnovel double-point modified 1,25(OH)2D2 analogs, PRI-5201 andPRI-5202, on human AML cells in comparison with their directprecursors (PRI-1906 and PRI-1907, respectively) and 1,25(OH)2D3.These experiments were carried out using three AML cell linesrepresenting different stages of myeloid differentiation: HL60(myeloblastic leukemia, consistent with the AML-M2 subtype),U937 (myelomonocytic leukemia; AML-M4) and MOLM-13(monocytic leukemia; AML-M5a). The extent of myeloid differen-tiation was assessed by bivariate analysis of surface expression ofthe specific monocytic marker CD14 and the general myeloidmarker CD11b (Suppl. Fig. 1; Suppl. Table 1). Treatment with each

Table 1Comparison of prodifferentiation potencies of different VDDs in AML cell lines.

Vitamin D derivatives HL60 U937 MOLM-13

1,25(OH)2D3 12.86 � 4.24 4.72 � 1.01 0.93 � 0.21PRI-1906 2.67 � 0.97 2.89 � 0.77 0.97 � 0.19PRI-1907 0.44 � 0.12 0.48 � 0.15 0.16 � 0.05PRI-5201 0.89 � 0.09* 0.59 � 0.09* 0.34 � 0.09*

PRI-5202 0.26 � 0.03** 0.32 � 0.02 0.07 � 0.01**

EC50 values (nM) were calculated by non-linear regression analysis of the dose-response curves for the CD14+/CD11b+ double-positive cell population (Fig. 2A). Thedata are the means � SD.

* p < 0.05 vs. PRI-1906.** p < 0.05 vs. PRI-1907.

Please cite this article in press as: M. Nachliely, et al., Novel analogs of 1,2efficient inducers of differentiation in human acute myeloid leukemia cejsbmb.2015.09.014

VDD for 96 h resulted in a dose-dependent increase in cell surfacelevels of CD14 and CD11b (Suppl. Fig. 2) as well as in the percentageof cells expressing both myeloid antigens, i.e., CD14+/CD11b+

double-positive cells (Fig. 2A; Suppl. Fig. 1; Suppl. Table 1). All thecell lines tested demonstrated a similar order of VDD potencies(PRI-5202 > PRI-1907 > PRI-5201 > PRI-1906 � 1,25(OH)2D3), as de-termined for the CD14+/CD11b+ cells (Table 1). Comparison of theEC50 values demonstrates that the potencies of PRI-5201 and PRI-5202 were higher relative to that of PRI-1906 and PRI-1907,respectively (Table 1). The difference between the potencies of PRI-5201 and PRI-1906 was more pronounced (�3–5-fold) comparedto that between PRI-5202 and PRI-1907 (�1.5–2.0-fold). Impor-tantly, in HL60 cells, PRI-5201 and PRI-5202 were about 15- and50-fold more potent than 1,25(OH)2D3, respectively. In U937 cells,this difference was about 8- and 15-fold and in MOLM-13 cells,about 3- and 13-fold, respectively.

The prodifferentiation effects of PRI-1906 and PRI-1907 havebeen extensively studied in several AML cell lines and patient-derived leukemic blasts [23,28], and were shown to be morepronounced than those of 1,25(OH)2D3. The data presented hereindicate that the ability of the new 1,25(OH)2D2 analogs to inducemyeloid differentiation is even greater compared to the abovecompounds.

3.2. Effects of 1,25(OH)2D3 and 1,25(OH)2D2 analogs on the growth ofAML cells

It has been well documented that 1,25(OH)2D3-induceddifferentiation of AML cells is accompanied with G1 cell cyclearrest, although the mechanisms underlying the two effects appearto be different (e.g., [29]). We, therefore, examined whether 1,25(OH)2D2 analogs can affect cell proliferation and/or viability. To thisend, following incubations with the VDDs for 96 h viable and deadcells were enumerated using the trypan blue exclusion assay. Wefound that while all the VDDs strongly induced myeloiddifferentiation in all the cell lines tested, HL60 and U937 cellsdisplayed only a modest reduction in cell numbers irrespective ofthe compound used (Fig. 2B), and without any change in cellviability (Suppl. Fig. 3). On the other hand, both 1,25(OH)2D3 andthe analogs were capable of markedly inhibiting the growth ofMOLM-13 cells (Fig. 2B), which was accompanied by a moderatedecrease in cell viability (Suppl. Fig. 3). Interestingly, the order ofantiproliferative activities of different VDDs in MOLM-13 cells wassimilar to that observed in the cell differentiation assays, i.e., PRI-5202 (IC50 = 0.04 nM) > PRI-1907 (IC50 = 0.11 nM) > PRI-5201 (IC50 =0.14 nM) > PRI-1906 (IC50 = 0.59 nM) = 1,25(OH)2D3 (IC50 = 0.57nM).

The reason for the marked difference in susceptibility of thethree AML cell lines to the growth-inhibiting effects of VDDs isintriguing, particularly, since in contrast to HL60 and U937 cells,the MOLM-13 cell line expresses wild-type p53, a multi-functionaltranscription factor that regulates DNA repair, cell proliferation,cell cycle and apoptosis. Although we have previously shown thatp53 status does not determine the differentiation-related G1 cellcycle arrest induced by 1,25(OH)2D3 in AML cells [30], the role ofthis transcription factor in the regulation of myeloid leukemia cellgrowth by distinct VDDs remains to be investigated.

3.3. Enhancement of the antileukemic effects of 1,25(OH)2D2 analogsby carnosic acid

Potentiation of the antileukemic activity of 1,25(OH)2D3 andsome of its analogs by plant polyphenols has been welldocumented (e.g., [16,18,20]); however, whether these botanicalscan cooperate with the 1,25-dihydroxyvitamin D2 analogs exam-ined here has not yet been investigated. We, therefore, compared

5-dihydroxyvitamin D2 combined with a plant polyphenol as highlylls, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.

Fig. 3. Carnosic acid enhances differentiation-inducing effects of 1,25-dihydroxyvitamin D2 analogs and inhibits proliferation in a cell type-specific manner. (A) Thepercentage of CD14+/CD11b+ cells was measured by flow cytometry. (B) The number of viable cells was determined by the trypan blue exclusion assay. The data are themeans � SD (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. the sum of the effects of single agents (A); and *, p < 0.05; **, p < 0.01 vs. VDD alone (B).

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the extent of the differentiation and proliferation of HL60,U937 and MOLM-13 cells treated for 96 h with low concentrationsof VDDs (0.25–1 nM), alone and in the presence of 10 mM carnosicacid. As shown in Fig. 3A, the untreated cell samples contained onlya marginal percentage of the more mature CD14+/CD11b+ cells andcarnosic acid alone had only a marginal effect on cell differentia-tion. However, combined treatments resulted in synergisticallyenhanced differentiation effects of 1,25(OH)2D3 and analogs in allthe cell lines tested, which occurred in both the VDD- and cell type-dependent manner. Similar results were obtained when the

Fig. 4. Differential effects of 1,25(OH)2D3 and 1,25-dihydroxyvitamin D2 analogs, and theiU937 cells. (A) Western blot assay following cell treatment with 10 mM carnosic acid arepresentative of 3 similar experiments is shown. (B and C) Cells transiently transfected w24 h. The relative VDRE � 6-Luc activity (means � SD) was calculated from the data of 3 in

Please cite this article in press as: M. Nachliely, et al., Novel analogs of 1,2efficient inducers of differentiation in human acute myeloid leukemia cejsbmb.2015.09.014

expression of CD14 and CD11b was analyzed separately (datanot shown). Enumeration of cells in these experiments demon-strated that carnosic acid alone modestly reduced the numbers ofHL60 cells but produced a more pronounced growth inhibition inthe U937 and, particularly, MOLM-13 cell lines. Co-incubation withVDDs either did not further enhance this inhibition or producedslightly stronger inhibitory effects compared to the polyphenolalone (Fig. 3B).

These results indicate that the 1,25(OH)2D2 analogs testedretain the ability of 1,25(OH)2D3 to synergistically cooperate with

r combinations with carnosic acid on VDR protein levels and VDRE transactivation innd/or 1 nM VDDs, for 48 h. Relative VDR/calregulin (CLRG) ratios are indicated. Aith VDRE � 6-Luc and Renilla plasmids, and incubated with the indicated agents fordividual experiments performed in quadruplicate. *, p < 0.05, vs. 1,25(OH)2D3 alone.

5-dihydroxyvitamin D2 combined with a plant polyphenol as highlylls, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.

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carnosic acid and, possibly, with other plant polyphenols. Thisfinding may have potential translational significance since we havepreviously reported that some low-calcemic analogs of 1,25(OH)2D3 and carnosic acid-rich rosemary extract synergisticallyretarded AML progression in mice without induction of hypercal-cemia [21,22]. Importantly, several recent clinical studies in whichVDDs were combined with differentiation-enhancing agentsshowed promising outcomes in patients with AML and myelodys-plastic syndrome (MDS). For instance, in a retrospective case-control study, median survival of elderly patients treated with 25-hydroxyvitamin D3 combined with the iron-chelating agentdeferasirox was significantly increased in comparison with thepatients receiving best supportive care [31]. In another study, a 4-year maintenance treatment with low-dose chemotherapy com-bined with 1,25(OH)2D3 and 13-cis retinoic acid in elderly patientswith AML and MDS resulted in a lower relapse incidence andlonger disease-free and overall survival compared to the patientswho did not receive maintenance therapy [32].

3.4. Effects of VDDs and carnosic on the vitamin D receptor levels andactivity in AML cells

Carnosic acid has been shown to modulate a wide array ofcellular regulatory processes, including signaling kinase pathways,redox status and cell cycle control (reviewed in [33]). We havepreviously reported that the enhancing effect of carnosic acid on1,25(OH)2D3-induced differentiation of AML cells is associatedwith the activation of the Raf-MEK-ERK1/2 [34] and JNK-AP-1-Egr-1 [19] pathways. Importantly, the carnosic acid/1,25(OH)2D3

combination also markedly upregulated the vitamin D receptor(VDR) levels and transcriptional activity through the activation ofthe transcription factors Nrf2 and AP-1 [17]. Here, using theU937 cell line as the model, we found that treatment with carnosicacid or a low concentration of each VDD (1 nM) alone resulted in anincrease in VDR protein levels to the various extent (Fig. 4A). Underthese conditions not only 1,25(OH)2D3 but also the analogs (exceptfor PRI-5201) cooperated with the polyphenol to enhance VDRprotein expression (Fig. 4A). The reporter gene assays performed inU937 cells transiently transfected with the vitamin D responseelement (VDRE)-luciferase construct demonstrated that, asexpected [17], carnosic acid which alone was ineffective synergis-tically potentiated VDRE transactivation by 1 nM 1,25(OH)2D3

(Fig. 4B). Similar to 1,25(OH)2D3,1,25(OH)2D2 analogs were capableof activating VDRE. However, surprisingly, carnosic did notsignificantly alter this activation (Fig. 4B). Further examinationof this system revealed that while at 1 nM all the VDDs activatedVDRE at a similar magnitude, at higher concentrations 1,25(OH)2D3

was clearly more efficacious (Fig. 4C).The above data demonstrate the discrepancy between the

inferior, with respect to 1,25(OH)2D3, ability of 1,25(OH)2D2

analogs, particularly, PRI-5201 and PRI-5202 to promote VDREtransactivation (Fig. 4C) and their superior potency in inducing thedifferentiation of AML cells (Fig. 2A). Furthermore, the failure ofcarnosic acid to potentiate VDRE activation by the analogs (Fig. 4B)is inconsistent with the facts that the polyphenol did enhance theirability to increase VDR levels (Fig. 4A) and to induce celldifferentiation (Fig. 3A). These differences are intriguing andmay suggest that the mechanisms by which 1,25(OH)2D3 and 1,25(OH)2D2 analogs induce AML cells to differentiate are distinct atleast in some aspects. One possibility, supported by findingsreported by Carlberg and coauthors (e.g., [35,36]), is that theconformation of VDR modified by 1,25(OH)2D2 analogs may bedifferent from that of the 1,25(OH)2D3-bound VDR. This may resultin an enhanced selective transactivation by the analog-bound VDR/RXR complex of a subset of differentiation-related VDRE sequencesthat are different from the DR3-type VDRE present in the reporter

Please cite this article in press as: M. Nachliely, et al., Novel analogs of 1,2efficient inducers of differentiation in human acute myeloid leukemia cejsbmb.2015.09.014

construct used in this study. For instance, it was shown that a 100-fold higher antiproliferative potency of the 1,25(OH)2D3 analogEB1089, relative to 1,25(OH)2D3, in MCF-7 human breast cancercells was due to an increased ability of EB1089 to transactivate theeverted repeat (ER) ER9-type VDREs compared to DR3-typeelements [35].

4. Conclusion

The principal and novel finding of this study is that the doublepoint-modified 1,25(OH)2D2 analogs (PRI-5201 and PRI-5202) thathave been recently synthesized by our group [26] exhibited asuperior differentiation-inducing activity in a panel of AML celllines compared to both 1,25(OH)2D3 and their direct precursorsPRI-1906 and PRI-1907, respectively. This enhanced activity wasachieved most likely by combining the synthetic modifications inthe structure of the A-ring (19-nor) and in the side-chain(optimized length and unsaturation level) of PRI-1906 and PRI-1907. Moreover, we found that, similar to 1,25(OH)2D3, the analogswere capable to cooperate with carnosic acid to induce enhancedantileukemic effects concomitant with upregulation of VDRprotein levels. The results of the VDRE transactivation assaysuggest that the superior prodifferentiation effects of the analogs,as compared to 1,25(OH)2D3, may be due to enhanced analog-mediated activation of a specific subset of VDREs in thedifferentiation-related genes. These results provide the basis forfurther mechanistic and translational testing of the antileukemicactivity of novel 1,25-dihydroxyvitamin D2 analogs and theircombinations with plant polyphenols.

Conflict of interest

The authors confirm that there are no known conflicts ofinterest associated with this publication and there has been nosignificant financial support for this work that could haveinfluenced its outcome.

Acknowledgements

The authors thank Dr. L.P. Freedman (presently affiliated withthe Global Biological Standards Institute, Washington, DC, USA) forhis kind gift of the VDRE reporter construct, and Dr. G.P. Studzinski(Rutgers-New Jersey Medical School, Newark, NJ, USA) for hiscomments and helpful discussions. This work was supported bythe Israel Science Foundation grant 635/11 and by the PeopleProgram (Marie Curie Actions) of the EU FP7/2007-2013 under REAgrant 315902,Initial Training Network DECIDE.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.jsbmb.2015.09.014.

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